Proper cellular function and differentiation depends on intrinsic signals and extracellular environmental cues. These signals and cues vary over time and location in a developing organism (i.e., during embryogenesis), and remain important in developing and differentiating cells during post-natal growth and in a mature adult organism. Thus, in a general sense, the interplay of the dynamically changing set of intracellular dynamics (such as manifested by intrinsic chemical signaling and control of gene expression) and environmental influences (such as signals from adjacent cells) determine cellular activity. The cellular activity so determined is known to include cell migration, cell differentiation, and the manner a cell interacts with surrounding cells.
The use of stem cells and stem-cell-like cells of various types for cell replacement therapies, and for other cell-introduction-based therapies, is being actively pursued by a number of researchers. Embryonic stems cells from a blastocyst stage are frequently touted for their pluripotency—that is, their ability to differentiate into all cell types of the developing organism. Later-stage embryonic stem cells, and certain cells from generative areas of an adult organism, are identified as more specialized, multipotent stem cells. These cells include cells that are able to give rise to a succession of a more limited subset of mature end-stage differentiated cells of particular types or categories, such as hematopoietic, mesenchymal, or neuroectodermal end-stage differentiated cells. For example, a multipotent neural stem cell may give rise to one or more neuron cell types (i.e., cholinergic neuron, dopaminergic neuron, GABAergic neurons), which includes their specific cell classes (i.e., a basket cell or a chandelier cell for GABAergic neurons), and to non-neuron glial cells, such as astrocytes and dendrocytes.
Further along the path of differentiation are cells derived from multipotent stem cells. For example, derivatives of a localized, non-migrating neuroectodermal type stem cell may migrate but, compared to their multipotent parent, have more limited abilities to self-renew and to differentiate (See Stem Cell Biology, Marshak, Gardner & Gottlieb, Cold Spring Harbor Laboratory Press, 2001, particularly Chapter 18, p. 407). Some of these cells are referred to a neuron-restricted precursors (“NRPs”), based on their ability, under appropriate conditions, to differentiate into neurons. There is evidence that these NRPs have different subclasses, although this may reflect different characteristics of localized multipotent stem cells (Stem Cell Biology, Marshak et al., pp 418-419).
One advantage of use of mulitpotent and more committed cells further along in differentiation, compared to pluripotent embryonic stem cells, is the reduced possibility that some cells introduced into an organism from such source will form a tumor (Stem Cell Biology, Marshak et al., p. 407). However, a disadvantage of cells such as cell types developed from multipotent stem cells, for instance, embryonic progenitor cells, is that they are not amenable to ongoing cell culture. For instance, embryonic neural progenitor cells, which are able to differentiate into neurons and astrocytes, are reported to survive only one to two months in a cell culture.
Generally, it is known in the art that the lack of certain factors critical to differentiation will result in no or improper differentiation of a stem cell. Researchers also have demonstrated that certain factors may be added to a culture system comprising stem cells, such as embryonic stem cells, so that differentiation to a desired, stable end-stage differentiated cell proceeds. It also is known in the art to introduce and express a transcription factor gene, Nurr1, into embryonic stem cells, and then process the cells through a five-step differentiation method (Kim, Jong-Hoon et al., Dopamine Neurons Derived from Embryonic Stem Cells Function in an Animal Model of Parkinson's Disease, Nature, 418:50-56 (2002)), resulting in differentiated cells having features of dopaminergic cells. However, the starting cell for this was an embryonic stem cell, and the differentiation process through to a cell having the features of a dopaminergic neuron, requires substantial effort that includes the addition and control of endogenous factors. In addition, because the starting cell is an early-stage embryonic stem cell having pluripotency, there is a relatively higher risk that some cells implanted from this source will become tumerogenic.
Also, without being bound to a particular theory, it is believed that to the extent a particular method of differentiation results in a greater percentage of cells that are dedicated or predetermined to differentiate to a desired functional cell type (i.e., a cholinergic neuron), this reduces the chance of tumor formation after introduction of cells derived from such method. As disclosed herein, embodiments of the present method that utilize multipotent stem cells as the starting material provide an increased percentage of cells predisposed (i.e., biased) to or differentiated to a desired cell type. This is believed to provide for reduced risk of tumor formation equivalent to or superior to the use of more differentiated cells such as NRPs.
There are many possible applications for methods, compositions, and systems that provide for improved differentiation of stem cells to a desired functional, differentiated cell. For example, not to be limiting, millions of people suffer from deafness and balance defects caused by damage to inner ear hair cells (IEHCs), the primary sensory receptor cells for the auditory and vestibular system after exposure to loud noises, antibiotics, or antitumor drugs. Since IEHCs rarely regenerate in mammals, any damage to these organs is almost irreversible, precludes any recovery from hearing loss, and results in potentially devastating consequences. Current therapies utilizing artificial cochlear implants or hearing aids may partially improve but not sufficiently restore hearing. Therefore, cell therapy to replace the damaged IEHC may be one of the most promising venues today. In the past, IEHC production from progenitor cells from the vestibular sensory epithelium of the bullfrog {Cristobal, 1998 #28} and possible existence of IEHC progenitors in mammalian cochlea sensory epithelia {Kojima, 2004 #29} has been reported. However limited quantity of IEHC progenitor prevents clinical application of this type of cell to treat deafness. Thus novel technology to produce IEHCs from other cell sources is needed.
While stem cells are known to be the building blocks responsible for producing all of a body's cells, the specific differentiation process towards to IEHC linage is not clear. Embryonic stem cells transplanted into the inner ear of adult mice or embryonic chickens did not differentiate into IEHCs {Sakamoto, 2004 #19}. Neural stem cells (NSCs) grafted into the modiolus of cisplatin-treated cochleae of mice only differentiated into glial or neuronal cells within the cochleae {Tamura, 2004 #18}. In order to produce IEHCs from these stem cells, modification or direction of the cell fate decision may be needed.
Another possible application for methods, compositions, and systems of the present invention is biasing Human Neural Stem Cells (“HNSCs”) to differentiate to cholinergic neurons, or to cells having characteristics of cholinergic neurons. Such biasing would provide for an improved percentage of such stem cells in a culture vessel to differentiate to this desired end-stage nerve cell. Improvements to the percentage of cells that are known to be biased to differentiate to this end-stage neuron cell, or to cells having characteristics of a cholinergic neuron, may lead to improvements both in research and treatment technologies for diseases and conditions that involve degeneration or loss of function of cholinergic neurons. Alzheimer's disease is one example of a malady known to be associated with degeneration of the long-projecting axons of cholinergic neurons.
Thus, there is a need in the art to improve the compositions, methods and systems that provide biased and/or differentiated cells from stem cells or stem-cell-like cells. More particularly, a need exists to obtain a higher percentage of desired cells from a pre-implantation cell culture, such as starting from multipotent stem cells and obtaining a higher percentage of cells committed to differentiate to a specified type of functional nerve cell, such as cholinergic neurons or inner ear hair cells. The present invention addresses these needs.